The ability of proteins to change structure impacts upon many biological processes, including the evolution of new folds and functions from ancestral precursors, the regulation of allosteric systems, and the development of amyloid diseases. The long-term goal of this work is to understand how large-scale structural change is promoted or prevented by changes in amino-acid sequence. To achieve this goal we study pairs of groups of proteins which are related by common ancestry but do not share a common structure, and which have undergone changes in structure due to mutational processes at the sequence level. The proposed work involves two model systems: the Cro family of bacteriophage DNA-binding proteins and the salivary lipocalins from bloodsucking insects. Members of the Cro family differ by a replacement of 1-helical with 2-sheet secondary structure across half of the length of a DNA-binding domain, converting an all-1 structure to an 1+2 structure. Members of the salivary lipocalins differ by a rearrangement of two 2-strands within an eight- stranded 2-barrel structure. For the Cro family we have identified two members, Xfaso 1 and Pfl 6, which retain 40% identical sequences despite major differences in structure. For the lipocalin family, preliminary results indicate that we have also identified a pair of family members, triabin and TLL4, with recognizably similar sequences but very different structures. The specific goals of the proposed work are to a) use mutagenesis to elucidate the key sequence determinants which distinguish the all-1 and 1+2 folds in cro proteins, b) design structurally ambivalent sequences which are compatible with both the all-1 and 1+2 folds observed in the Cro proteins, c) investigate the role of conformational strain as a cause and/or consequence of structural switching in Cro, d) establish the lipocalin system and initiate mutagenesis and design studies to identify the basis of strand-swapping in the lipocalin 2- barrel. Methods to be used include site-directed mutagenesis, chimeragenesis, protein design, biophysical characterization methods such as circular dichroism and analytical ultracentrifugation, and high-resolution structure methods including NMR spectroscopy and X-ray crystallography. PUBLIC HEALTH RELEVANCE: The ability of protein molecules to undergo large changes in three-dimensional structure in response to mutations or changes in conditions is an important issue in regulation, neurodegenerative disease and evolution. A protein's structural properties are fundamentally determined by its amino-acid sequence, and our goal is to study certain proteins as models to determine what features of their sequences are important for promoting or preventing large-scale changes in structure.